Prenylated Flavonoids as Antioxidant and Melanin Inhibitors From Stingless Bee ( Wallacetrigona incisa ) Propolis

Abstract

Propolis from 4 stingless bees (Homotrigona apicalis, Wallacetrigona incisa, Tetragonula fuscobalteata, and Tetragonula fuscibasis) was investigated in the search for medicinal and cosmetic materials from tropical rainforest resources. Methanol extracts of the propolis were screened using antioxidant and antimelanogenesis assays (tyrosinase enzyme activity and melanin inhibitor in B16 melanoma). The extract of H. apicalis showed the strongest antioxidant activity, both in the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays, with half-maximal inhibitory concentration values of 0.72 ± 0.01 (mg/mL) and 0.26 ± 0.00 (mg/mL), respectively. The H. apicalis extract also displayed the strongest inhibition of tyrosinase (53% at 100 µg/mL). In the B16 melanoma cell assay, the W. incisa extract showed the strongest inhibition of melanin (21%) and was less cytotoxic. The W. incisa extract was fractioned to isolate the compounds with biological activities. Two prenylated flavonoids were obtained, named broussoflavonol F and glyasperin A. Both showed potent antioxidant activities, as well as inhibiting melanin in B16 melanoma, but not tyrosinase activity. These results indicated the potential of methanol extract of W. incisa to be developed for cosmetic material, but further experiments are needed to verify the function.

Melanin, the major pigment of human skin, is located in the basal layer of the epidermis and is secreted by melanocytes. Melanin is very important in the protection of human skin from harmful ultraviolet (UV) and sun rays. ¹ Melanogenesis produces a reactive oxygen species (ROS) and reactive oxidants, including hydrogen peroxide, which creates oxidative stress in the melanocytes. Some ROS scavengers and inhibitors inhibit UV-induced melanogenesis and antioxidants, like reduced glutathione, and ascorbic derivatives are applied for treating several skin problems such as depigmentation of hyperpigmented spots. Therefore, many compounds have been reported as free radical scavengers, antioxidants, and melanin inhibitors, such as vitamin C, arbutin, kojic acid, ellagic acid, tranexamic acid, potassium methoxysalicylate, and 5,5-dipropyl-biphenyl-2,2-diol, which are important to protect human skin from the damaging effects of UV radiation, such as hyperpigmentation. ^2,3

Stingless bees are a group of insects belonging to 5 genera (Dectylurina, Melipona, Meliponula, Lestrimelitta, and Trigona), which play an important role in plant pollination. ⁴ These genera also produce propolis; a mixture of beeswax, plant resins, and pollens which is used to seal their beehives. Stingless bees’ propolis is used as a traditional medicine to heal some diseases related to digestive and respiratory systems, female fertility, skin, visual disorders, and various other ailments. ^5,6 Scientifically, propolis from stingless bees has some biological functions such as α-glucosidase inhibition, ⁷ antiproliferative activity in human cancer cells, ⁸ cytotoxicity to head and neck cancer cell lines, ⁹ and antimicrobial, anti-inflammatory, cytotoxic, ¹⁰ and antioxidant activities. ^10,11 Evaluation of propolis from stingless bees as a melanin inhibitor has not been reported previously. In this study, our screening data results for the propolis extracts from 4 stingless bees, namely, Homotrigona. apicalis, Wallacetrigona incisa, Tetragonula fuscobalteata, and Tetragonula fuscibasis, lead us to focus on the isolation of the active antioxidant and melanin inhibitors.

Materials and Methods

Materials

Propolis from H. apicalis, W. incisa, T. fuscobalteata, and T. fuscibasis was collected in February 2013 at Mulawarman University Forest Education, Samarinda, East Kalimantan, Indonesia. ¹² The stingless bees were identified by Dr Syafrizal and deposited in the Biology Laboratory, Biology Department, Faculty of Mathematics and Sciences, Mulawarman University.

Extraction, Fractionation, and Isolation of the Active Compounds

The propolis was sliced into pieces and extracted with methanol (MeOH). The propolis of H. apicalis (21.18 g) was extracted with 100 mL MeOH, the propolis of W. incisa (50.04 g) with 200 mL MeOH, the propolis of T. fuscobalteata (50.65 g) with 200 mL MeOH, and the propolis of T. fuscibasis (50.42 g) with 200 mL MeOH at room temperature for 48 hours. From H. apicalis propolis, 2.82 g dry extract was obtained, from W. incisa 10.79 g, from T. fuscobalteata 13.82 g, and from T. fuscibasis 11.63 g. About 10.02 g of W. incisa propolis extract was fractionated by silica gel column chromatography (114.4 g Wakogel C-200, 4.0 × 40 cm) and eluted with n-hexane/ethyl acetate (EtOAc) in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10 (v/v) and EtOAc/MeOH in ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10 (v/v) to yield 33 fractions (F1–F33). F10 (160 mg) was used for isolating active compounds by preparative high-performance liquid chromatography (HPLC) (Inertsil Prep-ODS:20 mm i.d. x 250 mm) and elution with MeOH/water (H₂O) (0.1% trifluoroacetic acid), 85:15 (v/v), 5 mL/min. Five fractions were obtained, including F10-5-5 (9.4 mg) and F10-5-6 (12.9 mg). Based on nuclear magnetic resonance (NMR) (¹H, ¹³C, distortionless enhancement by polarization transfer [DEPT], heteronuclear multiple-quantum correlation spectroscopy [HSQC], and heteronuclear multiple-bond correlation spectroscopy [HMBC]) spectroscopic data, fractions F10-5-5 and F10-5-6 contained broussoflavonol F ¹³ and glyasperin A, ^14,15 respectively (Figure 1).

Figure 1

Isolated compounds (broussoflavonol F ¹³ [left]; glyasperin A ^14,15 [right]).

Radical Scavenging (DPPH) Assay

This assay was performed as described by Arung et al. ¹⁶ Five hundred microliters of 60 µM 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 467 µL of ethanol were used as working solutions. The positive control in this assay was kaempferol.

ABTS Radical Cation Decolorization Assay

The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay used was as described by Tanaka et al. ¹⁷ Five milliliters of 7 mM ABTS solution and 88 µL of 140 mM potassium persulfate were used as working solutions. The results were determined and expressed in terms of Trolox equivalent antioxidant capacity (TEAC, µg/mg). Kaempferol was used as a positive control.

Tyrosinase Enzyme Assay

The assay was performed as described by Arung et al. ¹⁶ L-3,4-dihydroxyphenylalanine (333 µL of a 2.5 mM solution) and 600 µL of 0.1 M phosphate buffer (pH 6.8) were used as working solutions in this assay. The positive control was kojic acid.

Determination of Melanin Content and Cell Viability on B16 Melanoma Cells

The B16 melanoma cells were purchased from RIKEN Cell Bank. Eagle’s Minimum Essential Medium, 10% (v/v) fetal bovine serum, and 0.09 mg/mL theophylline were used to maintain the cells in the incubator at 37°C with a humidified atmosphere of 5% carbon dioxide. This assay was for determining melanin content and cell viability as previously described by Arung et al. ¹⁶ The positive control in this assay was arbutin.

Results and Discussion

We screened propolis extracts from 4 stingless bee species using antioxidant and melanogenesis assays in order to evaluate the potential use of these products as a cosmetic agent. Table 1 shows that the extract of H. apicalis propolis produced the highest DPPH scavenging activity (half-maximal inhibitory concentration (IC₅₀) = 0.72 mg/mL). In the DPPH and ABTS assays, the extracts of H. apicalis, T. fuscobalteata, and W. incisa showed the same activity (IC₅₀ = 0.26 mg/mL) but were higher than for T. fuscibasis (IC₅₀ = 0.72 mg/mL). In this experiment, we used Trolox as a positive control for the comparison of these extracts as an antioxidant. Biological activities of propolis as antioxidants using DPPH and ABTS assays have been seldom reported. However, there are reports of the antioxidant properties of propolis extracts of the stingless bees Scaptotrigona depilis, Melipona quadrifasciata, ¹⁸ Melipona orbignyi, ¹⁹ Tetragonisca fiebrigi, ¹⁰ and Melipona fasciculata. ²⁰ Many compounds have been recorded as antioxidants, including flavonoids, ¹⁹ phytosterols, terpenes, tocopherol, ¹⁸ tannins, ²⁰ and phenolics. ^{10,18 -20}

Table 1

Antioxidants of Some Propolis Samples.

Propolis extracts	DPPH		ABTS
	IC50 (mg/mL)	TEAC (µg/mg)	IC50 (mg/mL)	TEAC (µg/mg)
Homotrigona apicalis	0.72 ± 0.01	78.0 ± 1.1	0.26 ± 0.00	116.1 ± 2.5
Tetragonula fuscibasis	31.1 a ± 0.01	34.5 ± 0.15	0.72 ± 0.01	74.8 ± 1.7
Tetragonula fuscobalteata	38.7 a ± 0.02	44.6 ± 0.4	0.26 ± 0.00	115.9 ± 0.01
Wallacetrigona incisa	42.4 a ± 0.00	49.5 ± 0.6	0.26 ± 0.00	102.6 ± 3.2

ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; TEAC, tocoferol equivalent antioxidant capacity.

^aAt 1000 µg/mL.

Next, we screened the propolis samples in melanogenesis assays for tyrosinase enzyme activity and melanin inhibition in B16 melanoma cells. Figure 2 shows the results of the tyrosinase activity of the 4 propolis samples at 100 µg/mL. The propolis of H. apicalis showed 53% inhibition of tyrosinase enzyme activity (IC₅₀ <100 µg/mL), while those of T. fuscibasis, W. incisa, and T. fuscobalteata were 36% (IC₅₀ >100 µg/mL), 26% (IC₅₀ >100 µg/mL), and 14% (IC₅₀ >100 µg/mL), respectively. In this experiment, kojic acid, a well-known tyrosinase inhibitor, ²¹ used as the positive control, produced 90% inhibition at 10 µg/mL (IC₅₀ <10 µg/mL). As far as we know, there has not been a report on the antityrosinase activity of stingless bee propolis extracts. Figure 3 shows the melanin inhibition in B16 melanoma cells of the 4 propolis at various concentrations (25, 50 and 75 µg/mL). In this assay, the high melanin inhibition and high viability of cells (represented by MTT) are preferred and potent for a cosmetic agent. The propolis extract of H. apicalis at the concentration of 50 µg/mL inhibited 53% of the melanin growth in B16 melanoma cells and 96% of viable cells, whereas that of T. fuscibasis inhibited 76% melanin growth in B16 melanoma cells and 90% of viable cells at the concentration of 25 µg/mL, and the propolis of T. fuscobalteata inhibited 81% of the melanin growth in B16 melanoma cells and 78% of viable cells at the concentration of 25 µg/mL. The propolis extract of W. incisa inhibited 79% melanin growth in B16 melanoma cells and 87% of viable cells at the concentration 25 µg/mL. Arbutin, a well-known melanin inhibitor and less cytotoxic ²² used as the positive control inhibited 72% melanin growth in B16 melanoma cells and 90% of viable cells at the concentration 100 µg/mL. To our knowledge, it has not been reported that stingless bee propolis extracts inhibit melanin growth in B16 melanoma cells with less cytotoxicity (high viability cells).

Figure 2

Effect of 4 stingless bees’ propolis extracts on tyrosinase activity (all values: mean ± standard deviation).

Figure 3

Effect of 4 stingless bees’ propolis extracts on melanin inhibition of B16 melanoma cells (all values: mean ± standard deviation). MTT was representative of cell viability. Melanin was representative of melanin content in cells.

Based on the results (Table 1, Figures 2 and 3), thin-layer chromatography (TLC) data (not shown), and high propolis production of stingless bees, ²³ we selected W. incisa propolis for further fractionation to isolate the active compounds. The 33 fractions collected were screened using DPPH and tyrosinase assays (data not shown). Based on the TLC and HPLC data, and the DPPH and tyrosinase assay results, attention was focused on fraction 10 for isolating the active compounds. Using chromatographic methods and NMR assignments (¹H, ¹³C, DEPT, HSQC, and HMBC), broussoflavonol F ¹³ and glyasperin A ^14,15 were identified. We then evaluated these compounds for their antioxidant and melanogenesis inhibition activities (Table 2 and Figure 4). In previous research, we reported the antioxidant (DPPH and ABTS assays) and melanogenesis activities of glyasperin A from the leaves of Macaranga pruinosa. ²⁴ In Table 2, glyasperin A (IC₅₀ = 443.0 ± 8.0 µM) is more active in the DPPH assay as an antioxidant than broussoflavonol F (IC₅₀ = 623.3 ± 0.2 µM), but kaempferol, which is well known as an antioxidant, was superior with an IC₅₀ of 23.8 ± 3.3 µM. In the ABTS assay, glyasperin A (IC₅₀ = 210.0 ± 2.7 µM) was also more active as an antioxidant than broussoflavonol F (IC₅₀ = 226.9 ± 0.0 µM); the IC₅₀ of kaempferol was 111.0 ± 1.6 µM. It seems that the positions (positions 6 and 8) of the prenylated moiety have an effect on the antioxidant activities, but this hypothesis needs to be verified. Kim et al ³ reported that antioxidants play an important role in regulating hyperpigmentation and melanogenesis.

Figure 4

Effect of broussoflavonol F on melanin inhibition of B16 melanoma cells (all values: mean ± standard deviation). MTT was representative of cell viability and melanin was representative of melanin content in cells.

Table 2

Antioxidant Activites of Broussoflavonol F and Glyasperin A.

Compound	DPPH	ABTS
	IC50(μM)	IC50(μM)
Broussoflavonol F	623.3 ± 0.2	226.9 ± 0.0
Glyasperin A	443.0 ± 8.0 a	210.0 ± 2.7 a
Kaempferol	23.8 ± 3.3 a	111.0 ± 1.6 a

ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; IC50, half-maximal inhibitory concentration.

^aData from Arung et al. ²²

Figure 4 shows that broussoflavonol F inhibited melanin growth in B16 melanoma cells (54%) but showed less cytotoxicity at a concentration of 6 μΜ, which was better than arbutin, the positive control. Broussoflavonol F was then tested in the tyrosinase enzyme assay but did not show any inhibition (data not shown). Broussoflavonol F and glyasperin A showed antioxidant (DPPH and ABTS assays) activity and inhibited melanin growth in B16 melanoma cells. Some researchers reported the biological function of these compounds as antiplatelet, ²⁵ cytotoxic against P-388 cells, ²⁶ cytotoxic in some cancer cells, ^27,28 and xanthine oxidase inhibitors. ²⁹ To our knowledge, this is the first report about broussoflavonol F as an antioxidant and melanin inhibitor in B16 melanoma cells.

In summary, broussoflavonol F and glyasperin A could be used as cosmetic materials, especially as a skin whitening agent by inhibiting melanin growth and with antioxidant capacity. It still needs other experiments to be addressed for efficacy and safety in human use.